Extraction and purification method of active constituents from stem of lonicera japonica thunb., its usage for anti-inflammatory and analgesic drug

ABSTRACT

Disclosed is a method for extracting and purifying active constituents from honeysuckle ( Lonicera japonica  Thunb.) and its use. More particularly, this invention relates to an extraction and purification method of active constituents including sweroside from for honeysuckle stem (stem of honeysuckle where leaves are removed) by removing tannins, hardly soluble flavonoids, saponins, and the like. Thus obtained active constituents have better anti-inflammatory and analgesic effect, are safer and more stable than the conventional active constituents obtained from honeysuckle flower or honeysuckle leaves, and include sweroside which is an effective active ingredient of anti-inflammatory and analgesic drug.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an extraction and purification methodof active constituents from honeysuckle (Lonicera japonica Thunb.) andits use, more particularly to an extraction and purification method ofactive constituents including sweroside from honeysuckle stem (stem ofhoneysuckle where leaves are removed) by removing tannins, hardlysoluble flavonoids, saponins, and the like. Thus obtained activeconstituents have better anti-inflammatory and analgesic effect, aresafer and more stable than the conventional active constituents obtainedfrom honeysuckle flower or honeysuckle leaves, and include swerosidewhich is an effective active ingredient of anti-inflammatory andanalgesic drug.

The honeysuckle (Lonicera japonica Thunb.) is a semi-evergreen vinyshrub that grows naturally at the feet of mountains or levees in therange of an altitude of 50 to 600 meters in Japan, China and Korea. Itsflower bud (honeysuckle flower) and stem (honeysuckle stem) are used asherbal medicines for promotion of urination, detoxification, hemostasis,blood purification, treatment of tumor, edema treatment, cold, diarrhea,emesis, and the like [Illustrated Book of Korean Flora, Chang-Bok Lee,709, 1989, Hangmun Publishing Co., Seoul; Standard for Herbs (HerbMedicines) Not Covered by Korean Pharmacopoeia, Hyung-Joon Chi, Sang-InLee, 87, 305, 1988, Korea Medical Index Co., Seoul; Resource Plants inKorea, Tae-Kyung Kim, vol. 4, 148-149, 1996, SNU Press, Seoul]. Also,many traditional Chinese medicinal books including Sasang ConstitutionalMedicine and Gwangje Bigeup teach that it is good for treating a varietyof inflammatory abscesses inside and outside human bodies [SasangConstitutional Medicine for Chosun People, Yenben Chosun MedicalInstitute, 276, 1991, Yeogang Publishing Co., Seoul; Gwangje Bigeup,Kyung-Hwa Lee, 349-351, 1991, Yeogang Publishing Co., Seoul]. It haslong been used as a folk remedy for treatment of upper respiratoryinfections such as cold, tonsillitis and neuralgia for itsanti-inflammatory and analgesic activity. Recently, the inflammatory andanalgesic activity of honeysuckle has been proved through a variety ofexperimental animal models, and its effective physiologically activeconstituents have been isolated and reported to the academic circle[Development of the vegetable anti-inflammatory medicine: Comparison ofanti-inflammatory and analgesic actions of the honeysuckle extract,Song-Jin Lee et al., Korean Journal of Pharmacognosy, 363-367, 25, 1994;Flavonoids from the aerial parts of Lonicera japonica, Son et al., Arch.Pharm. Res., 365-370, 15, 1992; Antiinflammatory activity of Lonicerajaponica, Lee et al., Phytother. Res., 445-447, 12, 1998; Triterpenoidsaponins from the aerial parts of Lonicera japonica, Son et al.,Phytochem., 1005-1008, 35, 1994 ; Anti-inflammatory activity of themajor constituents of Lonicera japonica, Lee et al., Arch. Pharm. Res.,133-135, 18, 1995].

2. Description of the Related Art

To date, hydrolyzable tannins such as caffeoylquinic acid, methylcaffeate, chlorogenic acid and iso-chlorogenic acid, and iridoidglycosides such as loganin, sweroside, vogeloside and epi-vogeloside areknown as effective active constituents contained in honeysuckle stem.Most conventional researches have been centered on honeysuckle flowerand honeysuckle leaves. It should be noted that honeysuckle stem has adifferent distribution of constituents from honeysuckle leaves orhoneysuckle flower. That is, unlike honeysuckle stem, major constituentsof honeysuckle leaves or honeysuckle flower are flavonoids, such aslonicerin, rhoifolin and ochnaflavon, triterpene saponins havinghederagenin or oleanolic acid as non-sugar constituent, and varioushydrolyzable tannins.

These constituents are hard to be prepared into an injection. If theinjection is rich in polymer tannins, they may bind to otherconstituents thus resulting in coprecipitation, and may bind to serumproteins in the blood to form hardly soluble precipitates, which can bea cause of angiostenosis. In addition, since flavonoids included inhoneysuckle in general are insoluble to water, a fairly large amount oforganic solvent or other agents which mediate dissolution are necessaryto dissolve them to the level higher than the effective concentration.Also, active constituents rich in flavonoids are extremely insoluble inphysiological salt solution for injection and may become unstable ifstored for a long time in an alkaline buffer solution. Lastly, saponinsobtained from honeysuckle, particularly monodesmosides, are known tohave strong hemolysis properties. Therefore, they cannot be injecteddirectly into the vein without purification [Studies on the saponins ofLonicera japonica Thunb., Kawai et al., Chem. Pharm. Bull., 4769-4775,36(12), 1988]. Honeysuckle leaves and honeysuckle flower are greater intannins and hardly soluble flavonoids than honeysuckle stem. In theacute toxicity test for injections, they confer more toxicity thanhoneysuckle stem even at low contents, and provide poor analgesic andanti-inflammatory effect.

Lonicera japonica, Swertia japonica, Gentiana scabra, Gentiana triflora,Gentiana manshurica, Gentiana rigescens, and Gentiana rigescens French.var. stictantha Marquand have been used for alleviation of fever ordetoxification for many years. However, it has not been clearlyunderstood which constituents offer such effects, and most of theresearches have been concentrated on identifying the activities ofloganin, the major medicinal ingredient of honeysuckle stem [J. Nat.Prod., 54(4), 1102˜1104, 1991: Planta Med., 60, 232˜234, 1994:Phytotherapy Res., 12, 405-408, 1998].

Further, sweroside has been known as effective only in liver protectionand inhibition of bacterial activity [J. Ethnopharmacol., 42, 183-191,1994: Chem. Pharm. Bull., 45(11), 1823-1827, 1997: Yakugaku Zasshi,102(8), 755-759, 1982], and its anti-inflammatory and analgesic effecthas not been reported so far.

SUMMARY OF THE INVENTION

The present inventors have identified that active constituents ofhoneysuckle stem (stem of honeysuckle where leaves are removed), fromwhich tannins, hardly soluble flavonoids, saponins, and the like havebeen removed, and sweroside, the effective active ingredient of theactive constituents, have superior anti-inflammatory and analgesiceffect.

Accordingly, it is an object of the present invention to provide apreparation method for preparing active constituents from honeysucklestem, which have superior anti-inflammatory and analgesic activity,safety and stability.

It is another object of the present invention to provide ananti-inflammatory and analgesic drug comprising the active constituents.

It is still another object of the present invention to provide ananti-inflammatory and analgesic drug comprising sweroside.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graph that shows medicinal effects of the activeconstituents depending on their contents.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a method for preparing activeconstituents from honeysuckle stem, which have superioranti-inflammatory and analgesic activity, safety and stability.

The present invention also provides an anti-inflammatory and analgesicdrug comprising the active constituents.

The present invention also provides an anti-inflammatory and analgesicdrug comprising sweroside.

Hereunder is given a more detailed description of the present invention.

The present invention relates to a method extracting and purifyingactive constituents and sweroside from honeysuckle by removing tannins,hardly soluble flavonoids, saponins, and the like. Thus obtained activeconstituents have significantly better anti-inflammatory and analgesiceffect, are safer and more stable than the conventional activeconstituents obtained from honeysuckle flower or honeysuckle leaves, andalso include sweroside which is an effective active ingredient ofanti-inflammatory and analgesic drug.

Active constituents and sweroside are extracted from honeysuckle stemand purified by the following method.

A honeysuckle stem sample is reflux-extracted with about 7 to 10 volumesof distilled water for 2 to 3 hours, and then filtered. The residue iscollected and reflux-extracted with about 5 to 7 volumes of distilledwater for 2 to 3 hours. Thus obtained liquid is filtered and combinedwith the above filtrate, concentrated under reduced pressure, andfiltered again, so that its volume becomes about 1 to 3 times (v/w) withreference to the herb weight. In extracting with distilled water, ifdistilled water is used too little, stirring becomes difficult and theextraction efficiency decreases because the solubility of the extractbecomes poor. In contrast, if distilled water is used excessively, itrequires more time and cost. Then, equivalent water-saturated low gradealcohol is added and stirring is carried out for about 10 to 20 minutesat 30 to 50 rpm. After the layers are separated, the water-saturated lowgrade alcohol layer is filtered and concentrated under reduced pressureto obtain primary active constituents. Here, the water-saturated lowgrade alcohol to be used is prepared by adding distilled water to lowgrade alcohol, such as propyl alcohol and butyl alcohol, and stirringfollowed by sedimentation. The process of layer separation is carriedout for 2 to 3 times. In obtaining the low grade alcohol solventfraction, if low grade alcohol is used too little, the purificationefficiency decreases, thus the extraction yield and the effectiveingredient content decrease. In contrast, if low grade alcohol is usedtoo much, it results in increase in cost. Accordingly, it is recommendedto use 1 to 3 volumes (v/w) of low grade alcohol with reference to theherb weight.

A column chromatography is carried out for the primary activeconstituents using polyamide resin, polyvinylpyrrolidone resin, and thelike in order to remove unwanted materials and detect effectiveingredient. The filler is used in 1 to 10 volumes (w/w) of thewater-saturated low grade alcohol layer. Two three volumes of 50% (v/v)methanol and methanol to the filler volume are eluted, and then thedistilled water solvent is eluted by step-gradient method. The secondaryactive constituents obtained by eluting the active constituents withdistilled water had much less aromatic organic acids, tannins andflavonoids, and showed better medicinal effect, significantly reducedtoxicity, increased solubility and improved blood stability. A columnchromatography is carried out again for the secondary activeconstituents using ODS (octadecylsilane) resin. Starting from a 10%(v/v) methanol, 2 to 3 volumes of solvent to the resin volume is elutedby the step-gradient method while increasing the methanol content by 10%(v/v). The polyamide resin or polyvinylpyrrolidone resin is used in 20to 50 volumes for the weight of the purified primary activeconstituents. The active constituents obtained by eluting 20 to 30%(v/v) methanol showed the best anti-inflammatory and analgesic effect.Analysis of the active constituents revealed that iridoid substances,such as sweroside and loganin are major active ingredients. Thesweroside content was 15.1 to 72.1 wt %, and the loganin content was13.9 to 41.4 wt %.

The active constituents obtained by eluting 20 to 30% (v/v) methanol hasthe highest sweroside content. Another column chromatography was carriedout for these active constituents to separate sweroside represented bythe following Chemical Formula 1:

For thus obtained active constituents and sweroside, an arachidonic acidinduced ear edema test and a croton oil induced ear edema test werecarried out to determine the anti-inflammatory effect. And, an aceticacid induced writhing test was carried out to measure the analgesiceffect. As a result, they were found to have much superior inflammatoryand analgesic activities than the conventional active constituentsobtained from honeysuckle flower or honeysuckle leaves.

The sweroside obtained by the present invention can be prepared intotreatment drugs by the methods known in the pharmaceutical circle. And,it can be administered orally or parenterally alone or along with apharmaceutically acceptable carrier, a forming agent, a diluent, etc.Particularly, it can be prepared in the form of powder, granule, tablet,capsule, syrup, skin ointment or injection drug.

The human dosage of the active constituents or sweroside of the presentinvention can be selected considering the absorptivity of the activeingredient in the body, inactivation rate and excretion rate, age, sexand physical status of the subject, severity of the disease to betreated, and so forth. Preferably, the active constituents or swerosideare administered from 1 to 200 mg a day for an adult. The drug may beadministered according to specialized method, if required by theexperts' opinion. The drug may be administered several times a day,preferably 1 to 3 times a day, at regular intervals. The drugcomposition may be administered orally or non-orally. When the drug isto be administered parenterally, it can be administered through veins,muscles, rectum or skin.

Because the active constituents of honeysuckle stem have superioranti-inflammatory and analgesic effect and desirable solubility, acutetoxicity and blood stability, they are well suited for the injectiondrug.

Hereinafter, the present invention is described in more detail throughthe following examples. However, the following Examples are only for theunderstanding of the present invention, and they shall not be construedas limiting the scope of the present invention.

EXAMPLES Example 1 Medicinal Effects of Each Part of Honeysuckle

Samples of the whole body of honeysuckle (honeysuckle with stem andleaves), honeysuckle leaves and honeysuckle stem were taken atYeongcheon, Gyeongsangbuk-do, Korea in July 1999. The samples were driedin the shade and reflux-extracted with 7 volumes of distilled water for2.5 hours, and then filtered. The residue was then collected andreflux-extracted with 7 volumes of distilled water for 2.5 hours. Thusobtained liquid was filtered and combined with the above filtrate,concentrated under reduced pressure, and filtered again, so that itsvolume became about 2 times (v/w) with reference to the herb weight.Then, equal volume of water-saturated n-butyl alcohol was added and themixture was stirred for 15 minutes at about 30 rpm. After the layerswere separated, the alcohol layer was filtered and concentrated underreduced pressure to obtain primary active constituents. Then, a columnchromatography was carried out for obtaining the purified fraction usingpolyamide resin (CAS NO. 63428-83-1). The resin amount was 5 volumes ofthe sample. Two volumes of 50% (v/v) methanol and methanol were eluted,and then the distilled water solvent was eluted by step-gradient methodto obtain secondary active constituents. A croton oil induced ear edematest was carried out by administering the secondary active constituentsof whole body of honeysuckle, honeysuckle stem and honeysuckle leavesinto the tail veins of 6-week-old ICR mice (body weight: 20 to 30 g,n=6, SLC, Japan), which had been fasted for 4 hours. 15 minutes later,inflammation was induced with 2.5% croton oil. 4 hours later,thicknesses of left and right ears of the mice were measured using adial thickness gauge. The rate of inflammation was calculated by thefollowing Equation 1 and the result is shown in Table 1.Rate of inflammation (%)=[Thickness of inflamed (right) ear−Thickness ofnormal (left) ear]/[Thickness of normal ear]×100  Equation 1

TABLE 1 Classification Concentration (mg/kg) Rate of Inhibition (%)Whole body of 1 27.6 honeysuckle 3 29.7 10 34.8 25 33.6 50 32.2 100 30.4Honeysuckle stem 3 30.2 30 39.0 Honeysuckle leaves 3 19.6 30 21.9

As shown in Table 1, the active constituents of honeysuckle stem showedbest anti-inflammatory and analgesic activity.

Example 2 Comparison of Medicinal Effect of Active Constituents Obtainedfrom Honeysuckle Stem

A croton oil induced ear edema test was carried out as in Example 1.

Further, an arachidonic acid induced ear edema test was carried out byadministering drugs (marobiven, primary constituents of honeysucklestem, and secondary active constituents of honeysuckle stem) into thetail veins of 6-week-old ICR mice (body weight: 20 to 30 g, n=6, SLC,Japan), which had been fasted for 4 hours. Fifteen minutes later,inflammation was induced with 0.05% arachidonic acid. About 1 hourlater, thicknesses of left and right ears of the mice were measured, andthe rate of inhibition was calculated by Equation 1 and the result isshown in Table 2. TABLE 2 Rate of Inhibition (%) Classification Dose(mg/kg) CO* AA** Control group 50 21.0 NT (marobiven) 100 25.8 NT 20030.1 NT Primary active 50 35.6 NT constituents 100 36.2 NT 200 38.0 NTSecondary active 0.1 19.8 11.7 constituents 0.3 35.2 16.5 3 38.0 26.3 1036.6 32.4 30 37.1 48.7*Croton oil induced ear edema test result**Test result of arachidonic acid induced ear edemaNT: Not-tested

As shown in Table 2, the secondary active constituents obtained fromhoneysuckle stem included no compounds like aromatic organic acids,tannins and flavonoids, and had a greater content of active ingredientthan the primary active constituents.

Example 3 Comparison of Medicinal Effects of Active ConstituentsObtained from Honeysuckle Stem

The procedure of Example 1 was carried out by replacing polyamide resinwith polyvinylpyrrolidone resin (CAS NO. 25249-54-1). TABLE 3 Rate ofInhibition (%) Classification Dose (mg/kg) CO* AA** Control group 5020.2 NT (marobiven) 100 24.1 NT 200 31.2 NT Primary active 50 33.2 NTconstituents 100 35.1 NT 200 37.5 NT Secondary active 0.1 20.1 10.3constituents 0.3 34.1 16.7 3 39.7 24.9 10 35.6 33.3 30 36.4 49.6*Croton oil induced ear edema test result**Test result of arachidonic acid induced ear edemaNT: Not-tested

Example 4 Preparation of Final Active Constituents and Medicinal EffectTest

The secondary active constituents of honeysuckle stem prepared inExample 1 were concentrated under reduced pressure to obtain a powder.Another column chromatography was carried out for the powder using ODSresin (YMC*GEL ODS-A 12 nm, S-150 m or ODS-AM 12 nm, S-50 m or ODS-AQ 12nm, S-50 m). Three volumes of resin was used and 20% (v/v) methanol waseluted to obtain the final active constituents.

A croton oil induced ear edema test was carried out as in Example 1.

Further, an acetic acid induced writhing test was carried out byadministering drugs (marobiven and the final active constituents ofhoneysuckle stem) into the tail veins of ICR mice (body weight: 20 to 30g, n=8, SLC, Japan), which had been fasted for a day. Twenty minuteslater, 0.7% acetic acid was injected intraperioneally. Fifteen minuteslater, numbers of writhing for 10 minutes were counted to calculate therate of inhibiting inflammation. The result is shown in Table 4. TABLE 4Rate of Inhibition (%) Classification Dose (mg/kg) CO* AA** Controlgroup 1 6.6 37.9 (marobiven) 10 24.0 48.8 100 29.8 56.5 Final active 0.129.6 62.1 constituents 1 34.8 68.4 10 45.8 76.9*Croton oil induced ear edema test result**Test result of acetic acid induced writhingNT: Not-tested

Example 5 Contents of Active Ingredients and Medicinal Effect Test

From high performance liquid chromatography (HPLC) for the final activeconstituents prepared in Examples 1 and 4, the contents of activeingredient were identified: sweroside=15.1 to 72.1 wt % and loganin=13.9to 41.4 wt %. The sweroside and loganin contents of each sample aregiven in Table 5. The medicinal effect of the final active constituentsdepending on habitat and gathering time are given in Table 6. TABLE 5Contents of sweroside and loganin depending on habitat and time ofcollection Yeongcheon, Andong, Taishan, Xinxiang, Month Gyeongsangbuk-do(A) Gyeongsangbuk-do (B) China China January Lo: 34.6 Lo: 38.3 Lo: 38.2Lo: 31.4 Sw: 57.7 Sw: 55.4 Sw: 48.6 Sw: 59.2 Total: 92.3 Total: 93.7Total: 86.8 Total: 90.6 February Lo: 30.2 Lo: 32.3 Lo: 33.3 Lo: 28.6 Sw:64.1 Sw: 62.1 Sw: 54.2 Sw: 63.3 Total: 94.3 Total: 94.4 Total: 87.5Total: 91.9 March Lo: 22.7 Lo: 26.1 Lo: 25.8 Lo: 20.5 Sw: 70.2 Sw: 67.6Sw: 60.3 Sw: 72.1 Total: 92.9 Total: 93.7 Total: 86.1 Total: 92.6 AprilLo: 13.9 Lo: 15.9 Lo: 19.1 Lo: 14.2 Sw: 63.1 Sw: 60.1 Sw: 65.3 Sw: 69.7Total: 77.0 Total: 76.0 Total: 84.4 Total: 83.9 May Lo: 17.1 Lo: 14.2Lo: 18.7 Lo: 14.9 Sw: 52.3 Sw: 55.7 Sw: 66.2 Sw: 55.2 Total: 69.4 Total:69.9 Total: 84.9 Total: 70.1 June Lo: 19.4 Lo: 16.2 Lo: 23.5 Lo: 17.4Sw: 33.4 Sw: 38.2 Sw: 43.5 Sw: 42.6 Total: 52.8 Total: 54.4 Total: 67.0Total: 60.0 July Lo: 21.7 Lo: 25.3 Lo: 28.9 Lo: 22.4 Sw: 15.1 Sw: 17.5Sw: 29.8 Sw: 35.2 Total: 36.8 Total: 42.8 Total: 58.7 Total: 57.6 AugustLo: 23.5 Lo: 27.0 Lo: 30.1 Lo: 32.1 Sw: 29.4 Sw: 32.7 Sw: 35.9 Sw: 36.4Total: 52.9 Total: 59.7 Total: 66.0 Total: 68.5 September Lo: 25.3 Lo:30.1 Lo: 33.6 Lo: 39.2 Sw: 47.6 Sw: 45.3 Sw: 42.5 Sw: 40.2 Total: 72.9Total: 75.4 Total: 76.1 Total: 79.4 October Lo: 28.7 Lo: 32.2 Lo: 33.8Lo: 37..4 Sw: 52.2 Sw: 50.5 Sw: 48.2 Sw: 43.3 Total: 80.9 Total: 82.7Total: 82.0 Total: 80.7 November Lo: 39.6 Lo: 37.3 Lo: 38.0 Lo: 41.4 Sw:54.3 Sw: 58.3 Sw: 54.6 Sw: 45.6 Total: 93.9 Total: 95.6 Total: 92.6Total: 87.0 December Lo: 36.2 Lo: 33.7 Lo: 35.2 Lo: 40.2 Sw: 56.8 Sw:60.2 Sw: 58.3 Sw: 52.6 Total: 93.0 Total: 93.9 Total: 93.5 Total: 92.8Lo: Loganin,Sw: Sweroside

TABLE 6 Rate of Inhibition (%) Classification Contents (mg/kg) CO* AA**Control group 10 25.6 46.9 (marobiven) 100 28.8 56.8 Yeongcheon, July 137.2 70.2 Lo: 21.7, Sw: 15.1 10 46.3 75.1 Total: 36.8 Andong, July 140.3 72.2 Lo: 25.3, Sw: 17.5 10 51.2 84.3 Total: 42.8 Xinxiang, July 149.8 78.8 Lo: 22.4, Sw: 35.2 10 52.6 85.6 Total: 57.6 Taishan, September1 52.1 82.9 Lo: 33.6, Sw: 42.5 10 52.4 85.5 Total: 76.1 Andong, November1 53.4 84.6 Lo: 37.3, Sw: 58.3 10 53.2 87.7 Total: 95.6*Croton oil induced ear edema test result**Test result of acetic acid induced writhing

Example 6 Hemolysis Test for Final Active Constituents

Twenty mL of blood was collected from a rabbit's heart using a syringetreated with heparin. The blood was centrifuged for about 10 minutes.The supernatant was discarded and the residue was diluted with 10volumes of physiological salt solution for injection. After mixing bygentle shaking, 0.5 mL of the diluted blood and 0.5 mL of each drug ofExample 1 (final active constituents of honeysuckle stem and primaryactive constituents of honeysuckle stem, honeysuckle leaves and wholebody of honeysuckle) and the final active constituents of Example 4 wereput in a test tube (Physiological salt solution and distilled water(100% hemolysis) control groups were also prepared). The test tube wasincubated in a bath kept at 37° C. for 15 minutes, and then placed atroom temperature for 45 minutes. Lastly, after centrifuge at 2500 rpmfor 2 minutes, the upper layer was analyzed at 540 nm. TABLE 7 StandardClassification Hemolysis (%) deviation Control group (physiological saltsolution 0.73 0.17 for injection) Final active constituents, 5 × 10⁻⁴g/mL 0.69 0.10 Final active constituents, 1.5 × 10⁻³ g/mL 0.73 0.11Final active constituents, 5 × 10⁻³ g/mL 0.74 0.88 Honeysuckle stem, 5 ×10⁻³ g/mL 15.2 0.20 Honeysuckle leaves, 5 × 10⁻³ g/mL 35.1 0.15 Wholebody of honeysuckle, 5 ×10⁻³ g/mL 23.0 0.45

Example 7 Preparation of Sweroside

A column chromatography was carried out for the final activeconstituents prepared in Example 4 using octadecylsilane resin toseparate sweroside represented by the following Chemical Formula 1:

Example 8 Determination of Anti-inflammatory Effect of Sweroside

A croton oil induced ear edema test was carried out by administeringsweroside into the tail veins of 6-week-old ICR mice (body weight: 20 to30 g, n=6, SLC, Japan), which had been fasted for 4 hours. Fifteenminutes later, inflammation was induced with 2.5% croton oil. Four hourslater, thicknesses of left and right ears of the mice were measuredusing a dial thickness gauge. The rate of inflammation was calculated byEquation 1 and the result is shown in Table 8. The medicinal effect fororal administration is given in Table 9. TABLE 8 Classification Dose(mg/kg) Rate of Inhibition (%) Sweroside 0.1 49.8 1 56.2 10 65.7

TABLE 9 Classification Dose (mg/kg) Rate of Inhibition (%) Sweroside 131.9 10 43.1 100 57.8

Further, an arachidonic acid induced ear edema test was carried out byadministering sweroside into the tail veins of 6-week-old ICR mice (bodyweight: 20 to 30 g, n=6, SLC, Japan), which had been fasted for 4 hours.Fifteen minutes later, inflammation was induced with 0.05% arachidonicacid. One hour later, thickness of left and right ears of the mice wasmeasured, and the Rate of Inhibition was calculated by the Equation 1and the result is shown in Table 10. The medicinal effect for oraladministration is given in Table 9. TABLE 10 Classification Dose (mg/kg)Rate of Inhibition (%) Sweroside 0.1 48.3 1 55.1 10 69.1

TABLE 11 Classification Dose (mg/kg) Rate of Inhibition (%) Sweroside 129.0 10 34.7 100 45.7

Example 9 Determination of Analgesic Effect of Sweroside

An acetic acid induced writhing test was carried out by administeringsweroside into the tail veins of ICR mice (body weight: 20 to 30 g, n=8,SLC, Japan), which had been fasted for a day. Twenty minutes later, 0.7%acetic acid was injected intraperioneally. Fifteen minutes later, thenumber of writhing for 10 minutes were counted to calculate the rate ofinhibiting inflammation and the result is shown in Table 12. Themedicinal effect for oral administration is given in Table 13. TABLE 12Classification Dose (mg/kg) Rate of Inhibition (%) Sweroside 0.1 74.6 186.4 10 89.7

TABLE 13 Classification Dose (mg/kg) Rate of Inhibition (%) Sweroside 165.9 10 79.1 100 87.9

Example 10 Toxicity Test

The active constituents of honeysuckle stem and sweroside, in the amountof 1.0 g/kg, 1.5 g/kg and 2.0 g/kg, were administered through the tailveins of SD rats (body weight: 120 to 170 g, 5 male and female rats foreach administration dose, SLC, Japan), respectively, which had beenfasted for 4 hours. The rats were observed for 30 minutes at first, andthen observed at 30 minutes intervals with naked eyes. Death rate,general symptoms and weight change were observed for 2 weeks afteradministration. An autopsy was conducted to identify presence of anyabnormalities of organs.

The lethal dose of the active constituents of honeysuckle stem andsweroside was both over 5.0 g/kg for oral administration (no dead ratsobserved), and over 2.0 g/kg for intravenous injection (no dead ratsobserved). When 2.0 g/kg was intravenously injected, the rats showedincreased respiration counts and decreased activity for about 10minutes, which were restored soon. No other symptoms were observed, andthere was no change in weight due to administration. The autopsy resultshowed no abnormalities as is the case with the control groups.

A local toxicity test at the dose of 50, 100 and 150 mg/kg,respectively, showed no difference from the group administered withphysiological salt solution. No toxicity, such as tissue necrosis orinflammation, was observed.

Preparation Example 1 Preparation of Tablet

The active constituents of honeysuckle stem or sweroside was preparedinto a tablet with the following composition: Active ingredient 160 mgLight anhydrous silicic acid 20 mg Corn starch 87 mg Crystallinecellulose 72 mg Sodium starch glyconate 60 mg Magnesium stearate 6 mgTotal 672 mg

Preparation Example 2 Preparation of Syrup

The active constituents of honeysuckle stem or sweroside was preparedinto syrup with the following composition: Active ingredient 4,000 mgMethyl p-oxybenzoate (5% ethanol solution) 60 mg Propyl p-oxybenzoate(5% ethanol solution) 40 mg Sodium benzoate (5% solution) 100 mg Bananapowder (10% solution) 600 mg D-Sorbitol 140,000 mg Distilled water 196mL

Preparation Example 3 Preparation of Injection

The active constituents of honeysuckle stem or sweroside was preparedinto an injection with the following composition: Injection ampule:Active ingredient 20 mg Mannitol 60 mg Corresponding solventPhysiological salt solution 2000 mg sample: for injection Total 2080 mg

Preparation Example 4 Preparation of Injection

The active constituents of honeysuckle stem or sweroside was preparedinto an injection with the following composition:

Injection ampule: Active ingredient 50 mg KH₂(PO₄) 8.5 mg Physiologicalsalt solution for injection 3000 mg Total 3058.5 mg

Preparation Example 5 Preparation of Injection

The active constituents of honeysuckle stem or sweroside was preparedinto an injection with the following composition:

Injection ampule: Active ingredient 100 mg Mannitol 300 mg KH₂(PO₄) 17mg Physiological salt solution for injection 3000 mg Total 3417 mg

Preparation Example 6 Preparation of Ointment Drug

The active constituents of honeysuckle stem or sweroside was preparedinto an ointment drug the following composition: Active ingredient 5 gLiquid paraffin 10 g Hard lead 9 g Ethanol 8 g Sorbitan monooleate 2 gPolysorbate 4 g Propyl p-oxybenzoate 0.05 g Methyl p-oxybenzoate 0.1 gConcentrated glycerine 10 g Purified water Adequate

As described above, the active constituents of the present inventionobtained from honeysuckle stem have significantly increased solubilityand blood stability as compared to those of the conventional activeconstituents. Further, they are shown to have superior analgesic andanti-inflammatory effect, safety and stability. Also, sweroside of thepresent invention obtained from honeysuckle stem has very superiormedicinal effect and shows little toxicity, thus being very suitable foran anti-inflammatory and analgesic drug.

While the present invention has been described in detail with referenceto the preferred embodiments, those skilled in the art will appreciatethat various modifications and substitutions can be made thereto withoutdeparting from the spirit and scope of the present invention as setforth in the appended claims.

1. A preparation method of active constituents of honeysuckle stem,which comprises: (a) a step of reflux-extracting honeysuckle stem withwater, filtering it, carrying out layer separation by adding equivalentwater-saturated low grade alcohol to said filtrate, and concentrating itunder reduced pressure to obtain primary active constituents; and (b) astep of purifying the primary active constituents with polyamide resinor polyvinylpyrrolidone resin to obtain secondary active constituents,and purifying again with octadecylsilane resin to obtain final activeconstituents.
 2. The preparation method of active constituents ofhoneysuckle stem according to claim 1, wherein said water-saturated lowgrade alcohol is saturated propyl alcohol or butyl alcohol solution. 3.The preparation method of active constituents of honeysuckle stemaccording to claim 1, wherein said active constituents comprisesweroside and loganin as effective ingredient.
 4. The preparation methodof active constituents of honeysuckle stem according to claim 3, whereinsaid active constituents comprise 15.1 to 72.1 wt % of sweroside and13.9 to 41.4 wt % of loganin.
 5. An anti-inflammatory and analgesic drugwhich comprises said active constituents of honeysuckle stem prepared bythe method according to claim
 1. 6. The anti-inflammatory and analgesicdrug according to claim 5, wherein said active constituents comprisesweroside and loganin as effective ingredient.
 7. The anti-inflammatoryand analgesic drug according to claim 6, wherein said activeconstituents comprise 15.1 to 72.1 wt % of sweroside and 13.9 to 41.4 wt% of loganin.
 8. The anti-inflammatory and analgesic drug according toclaim 5, which is prepared in the form of tablet, powder, granule,capsule, syrup, skin ointment or injection.
 9. An anti-inflammatory andanalgesic drug which comprises sweroside represented by the followingChemical Formula 1: